Coenzyme B12 (aka, adenosylcobalamin, AdoCbl) biosynthesis is a major biosynthetic pathway (>25 genes) that is unique to prokaryotes. Given the complex chemistry required to assemble this coenzyme, studies of its biosynthesis offer an opportunity to learn complex enzymology, protein-protein interactions, multienzyme complex systems, and to investigate the integration of this pathway into the metabolic network. Comprehensive knowledege ofthe pathway and its components hat can ultimately be used in antimicrobial design and anti-cancer drug development. We will expand the multifaceted approach we have used for years to include metabolomics approaches to investigate the several aspects of coenzyme B12 biosynthesis that remain unresolved. We propose to do four things: i) to perform in-depth studies of the mechanism of function of the recently discovered, Fe-S-containing, oxygen-labile EutT corrinold adenosyltransferase, and to understand the role of the metal in EutT catalysis; ii) to Identify genes of Salmonella that encode enzymes that synthesize 5,6- dimethylbenzimidazole (DMB, the lower ligand base of AdoCbl), and alpha-ribazole, the DMB nucleoside in the absence ofthe two known base-activating enzymes. We have strong genetic evidence ofthe existence of these unprecedented functions; iii) to identify the genes encoding the enzymes of the anaerobic of DMB biosynthetic pathway found in many human pathogens, and study the function of their products in vitro and in vivo. The analysis of this pathway has remained unexplored; and iv) to analyze the metabolome of strains of Salmonella that have been engineered to accumulate B12 intermediates that have only been studied in vitro. We will continue to collaborate with structural biologists led by Ivan Rayment (UW-Madison), with transition- metal spectroscopists led by Thomas Brunold (UW-Madison), and with mass spectrometrists led by Shawn Campagna {U of Tennessee-Knoxville). We expect to discover new enzymes and pathways, to understand their mechanism of catalysis, with the ultimate goal of understanding the function of the predicted membrane-associated multienzyme complex in vivo and vitro. RELEVANCE (See instructions): Only prokaryotes make CoB12, and many of which are human pathogens. Precise knowledge ofthe biochemistry underpinning the pathway and an understanding ofthe structural properties ofthe enzymes involved, is critical to the design of antimicrobials that would have no side effects on the host and could be used to target disease-causing bacteria. The use of B12 bioconjugates to fight cancer is also very promising.